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United States Patent |
5,300,653
|
Nozaki
,   et al.
|
April 5, 1994
|
Separation method of amino acids
Abstract
A method is provided for the separation of at least one aromatic amino acid
from an aqueous solution of mixed amino acids including the aromatic amino
acid. The aqueous solution is brought into contact with a strongly acidic
gel-type cation exchange resin which has been converted into a salt with
an alkali metal or an alkaline earth metal, whereby the aromatic amino
acid is selectively sorbed by the cation exchange resin. The aromatic
amino acid thus sorbed can then be desorbed from the ion exchange resin,
preferably with water.
Inventors:
|
Nozaki; Shohei (Yokohama, JP);
Murata; Naohiro (Kamakura, JP);
Miyazaki; Kiyoo (Yotsukaido, JP)
|
Assignee:
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Mitsui Toatsu Chemicals, Incorporated (Tokyo, JP)
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Appl. No.:
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995959 |
Filed:
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December 23, 1992 |
Foreign Application Priority Data
Current U.S. Class: |
548/497; 548/496; 562/443; 562/445 |
Intern'l Class: |
C07D 209/20; C07C 229/36 |
Field of Search: |
548/497
562/443,445
|
References Cited
U.S. Patent Documents
3045026 | Jul., 1962 | Eisenbraun.
| |
4133753 | Jan., 1979 | Takeuchi et al. | 562/443.
|
4769474 | Sep., 1988 | Miyahara et al. | 548/497.
|
4910336 | Mar., 1990 | Goodman.
| |
4956471 | Sep., 1990 | Ito et al. | 562/554.
|
5030750 | Jul., 1991 | Kuzira et al. | 562/554.
|
Foreign Patent Documents |
0336818 | Oct., 1989 | EP.
| |
61-189266 | Aug., 1986 | JP | 548/497.
|
1145512 | Mar., 1969 | GB.
| |
Other References
Gutsche et al., Fundamentals of Organic Chemistry, Prentice-Hall, Englewood
Cliffs (1975) pp. 113-118.
Roberts et al., Basic Principles of Organic Chemistry, W. A. Benjamin
(1964) pp. 1006-1007.
Dougherty et al., J. Chromatog., vol. 42 pp. 415-416 (1969).
|
Primary Examiner: Dees; Jose G.
Assistant Examiner: Frazier; B.
Attorney, Agent or Firm: Burns, Doane, Swecker & Mathis
Parent Case Text
This application is a continuation of application Ser. No. 07/612,478,
filed Nov. 14, 1990 now abandoned.
Claims
What is claimed is:
1. A method for separating at least one aromatic amino acid having a
benzene nucleus from an aqueous solution of mixed amino acids including
said at least one aromatic amino acid, which comprises:
providing a strongly acidic gel-type cation exchange resin which is a
sulfonated product of a styrene-divinylbenzene copolymer and has been
converted into a salt with an alkali metal or an alkaline earth metal; and
bringing said aqueous solution into contact with said cation exchange
resin, whereby said at least one aromatic amino acid is selectively sorbed
by said cation exchange resin and wherein said sorbed amino acid is
desorable with water.
2. The method of claim 1, further comprising desorbing said at least one
aromatic amino acid from said cation exchange resin.
3. The method of claim 2, wherein said at least one amino acid is desorbed
with water from said cation exchange resin.
4. The method of claim 1, wherein said solution contains at least one
aliphatic amino acid.
5. The method of claim 1, wherein said cation exchange resin is packed in a
column and is used as a packed layer.
6. The method of claim 1, wherein said cation exchange resin is in the form
of a salt with an alkali metal selected from the group consisting of
lithium, sodium, potassium, rubidium and cesium.
7. The method of claim 1, wherein said cation exchange resin is in the form
of a salt with an alkaline earth metal selected from the group consisting
of beryllium, magnesium, calcium, strontium and barium.
8. The method of claim 1, wherein said at least one aromatic amino acid is
selected from the group consisting of tryptophan, phenylalanine and
tyrosine.
Description
BACKGROUND OF THE INVENTION
a) Field of the Invention
The present invention relates to a method for separating at least one amino
acid from an aqueous solution of mixed amino acids, which solution is
produced, for example, in an amino acid production process such as a
synthetic process, a fermentation process, an enzymatic process or a
proteolytic process.
b) Description of the Related Art
As adsorption-dependent separation methods for amino acids, ion exchange
methods have been known for many years. According to one example of these
ion exchange methods, treatment of an aqueous solution of various amino
acids with a strongly acidic ion exchange resin is effected by converting
the resin into the free form, namely, the H form, causing the resin to
adsorb the amino acids thereon and then eluting the amino acids with
aqueous ammonia. This method makes use of the fact that an amino acid is
an ampholyte, whereby the amino acids are once bonded to functional groups
of the ion exchange resin. Accordingly, the pH of the solution of the
amino acids is lowered to convert the amino acids into cations, followed
by the treatment with the ion exchange resin. The amino acids adsorbed on
the resin through ion bonding are then treated with aqueous ammonia as an
eluent to raise the pH, so that the cations of the amino acids are
subjected to ion exchange with ammonium ions and are hence eluted.
Further, the ion exchange resin can be regenerated into the H form with a
mineral acid such as hydrochloric acid or sulfuric acid and can then be
employed again for the adsorption of amino acids
For example, Japanese Patent Application Laid-Open No. 73050/1981 discloses
a method in which one or more aromatic amino acids in an aqueous solution,
an aqueous alcohol solution or an alcohol solution are purified using a
strongly acidic ion exchange resin of the macroporous type. Even in this
method, the ion exchange resin is converted into the H form and, after
ionic adsorption of the amino acids, the amino acids are eluted with an
aqueous ammonia solution.
Serious drawbacks of such ion exchange methods reside in the use of an acid
as a pH-adjusting agent or a regenerating agent and also in the use of
aqueous ammonia as an eluent. To practice the above ion exchange methods
on an industrial scale, many accompanying problems therefore arise For
example, the acid employed must eventually be neutralized and discharged
as an effluent from the system. On the other hand, ammonia used as an
eluent accompanies amino acids as target substances for the separation
Removal of ammonia is therefore indispensable to isolate the amino acids.
Further, to discharge the thus-removed ammonia out of the system, it is
necessary not only to neutralize the same but also to eliminate by a
certain method its nitrogen fraction which causes trouble from the
standpoint of environmental protection.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a method for efficiently
separating one or more specific amino acids from a solution, in which the
specific amino acids are contained along with impurities, without
developing the above-described drawbacks of the conventional methods.
In one aspect of the present invention, there is thus provided a method for
separating at least one aromatic amino acid from an aqueous solution of
mixed amino acids including said at least one aromatic amino acid. The
method comprises providing a strongly acidic gel-type cation exchange
resin which has been converted into a salt with an alkali metal or an
alkaline earth metal; and bringing said aqueous solution into contact with
said cation exchange resin, whereby said at least one aromatic amino acid
is selectively sorbed by said cation exchange resin.
According to the present invention, desorption of the amino acid thus
sorbed can be conducted with purified water without relying upon an acid
or an alkali. Salt-forming ions bonded to the ion exchange resin, such as
sodium ions, remain bonded throughout the treatment so that they are not
eluted. Accordingly, the resin can be used, as it is, for the next
separation treatment.
In the light of the chemical and technical common knowledge of those
skilled in the art, it is believed to be impossible to achieve selective
ion exchange separation of an amino acid by treating a solution of mixed
amino acids with an ion exchange resin which has been converted into an
alkali metal salt or alkaline earth metal salt form. Therefore, the
present invention can also be considered extremely surprising from this
viewpoint.
DETAILED DESCRIPTION OF THE INVENTION
Exemplary amino acids to which the present invention can be applied include
aromatic amino acids--such as tryptophan, phenylalanine and tyrosine, and
their alkyl derivatives, hydroxyl derivatives, alkoxyl derivatives and
acetylated derivatives--as well as substituted derivatives of these
aromatic amino acids Their specific examples include phenylglycine; 4-
hydroxy- and 4-methoxyphenylglycines; phenylalanine; 4-methyl-,
2,3-dimethyl-, 4-isopropyl-, 4-chloro-, 4-fluoro-, 2-bromo-, 4-nitro-,
4-amino-, 4-methoxy-, 4-acetoxy-, 3,4-methylene-, 3,4-dihydroxy,
2-hydroxy- and 4-mercapto-phenylalanines; tyrosine; thyroxine; thyronine;
phenylserine; kynurenine; tryptophan; 2-methyl-, 5-methyl-, 2-hydroxy- and
5-hydroxy-tryptophans; furylalanine; thienylalanine; naphthylalanine; and
pyridylalanine.
The solution to be treated in accordance with this invention contains, in a
dissolved form, at least one of the above aromatic amino acids and their
substituted derivatives. The solvent of the solution to be treated is
water or an aqueous solvent Impurities other than amino acids, said
impurities being generally mixed in during a production process of an
amino acid such as a synthetic process, a fermentation process, an
enzymatic process or a proteolytic process, do not provide problems in the
process of the present invention.
Illustrative aliphatic amino acids which may be contained in a solution to
be treated include glycine, alanine, valine, leucine, isoleucine, serine,
threonine, proline, methionine, arginine, histidine, lysine, aspartic
acid, and glutamic acid.
Sorption of these non-aromatic amino acids on a strongly acidic gel-type
cation exchange resin converted into an alkali metal salt or alkaline
earth metal salt type is far more difficult compared to aromatic amino
acids. One or more aromatic amino acids which are contained along with
non-aromatic amino acids and are to be separated can therefore be
specifically sorbed and separated by the resin when treated in accordance
with the present invention.
No particular limitation is imposed on the concentration range of each
aromatic amino acid in a solution which can be treated successfully by
applying the present invention, as long as the aromatic amino acid is
dissolved therein. However, the concentration may range, for example, from
0.01% to 20%, preferably 0.05% to 10% for tryptophan; from 0.01% to 25%,
preferably 0.05% to 15% for phenylalanine; and from 0.001% to 10%,
preferably 0.005% to 5% for tyrosine
No particular limitation is imposed on the concentration range of each
coexisting aliphatic amino acid in a solution which can be treated
successfully by applying the present invention, as long as the aliphatic
amino acid is dissolved therein. However, the concentration may range, for
example, from 0.01% to 80%, preferably 0.01% to 30%, more preferably 0.02%
to 25%. It may be 0.0I% to 50%, preferably 0.02% to 40% for serine; and
from 0.01% to 25%, preferably 0.02% to 20% for alanine.
As the strongly acidic gel-type ion exchange resin in the present
invention, a sulfonated product of a styrene-divinylbenzene copolymer is
used. Exemplary cation exchange resins of this sort are commercially
available under various trade names such as "Lewatit S100", "Lewatit
S109", "Lewatit MDS1368" and "Lewatit TSW40" (products of Bayer AG);
"DIAION SK1B" (product of Mitsubishi Chemical Industries, Ltd.); "Dowex
HCR-S", "Dowex 50WX1" and "Dowex 50WX2" (products of Dow Chemical
Company); and "Amberlite IR120" and "Amberlite IR122" (products of Rohm &
Haas Company).
To use these cation exchange resins for the objects of the present
invention, they must be in the form of an alkali metal salt or an alkaline
earth metal salt. If they are not commercially available in such a form,
it is necessary to convert an H-form ion exchange resin into such a salt
form. As a salt-forming element, one or more metals selected from Li, Na,
K, Rb, Cs, Be, Mg, Ca, Sr and Ba can be suitably employed. To convert an
ion exchange resin into the form of such an alkali metal salt or alkaline
earth metal salt, it is necessary, as known per se in the art, to bring an
H-form ion exchange resin into contact with an aqueous solution of an
alkali metal hydroxide such as sodium hydroxide or potassium hydroxide, an
alkali metal halide, an alkali metal sulfate, an alkaline earth metal
halide or an alkaline earth metal sulfate for the treatment of the former
with the latter.
A description will next be made of an exemplary sorption operation in this
invention, A column is packed with the above ion exchange resin which has
been converted into an alkali metal salt or alkaline earth metal salt
form. A solution to be treated, which contains an amino acid to be
separated, is passed as an upflow or downflow through the packed layer,
whereby the solution is brought into contact with the ion exchange resin
and is treated with the same resin.
The pH of the solution to be treated is preferably 2-12, more preferably
4-11.
Although no particular limitation is imposed on the flow velocity of the
solution upon passing it through the column, the flow velocity may
generally be in the range of from 0.1 hr.sup.-1 to 100 hr.sup.-1,
preferably from 0.3 hr.sup.-1 to 60 hr.sup.-1 in terms of space velocity.
To desorb the amino acid thus sorbed, the ion exchange resin with the amino
acid sorbed thereby is brought into water which is substantially free of
any acid or alkali. An organic solvent or a mixture of an organic solvent
and water can also be used if desired.
No particular limitation is imposed on the operation temperature for
practicing the above sorption and desorption. However, the operation may
generally be conducted at a temperature of from 0.degree. C. to
100.degree. C. The operation temperature at the time of sorption may be
same as or different from that at the time of desorption. Sorption at a
relatively low temperature is however advantageous as it results in a
higher sorption capacity. In addition, desorption at a higher temperature
is advantageous because the amount of a desorbing solvent can be reduced.
As an advantageous feature of the method of this invention, it is to be
noted that no special desorbing solvent is required in the above desorbing
operation.
In addition, the salt-forming element such as an alkali metal is always
fixed on the ion exchange resin. It is therefore another important feature
of the present invention that the salt-forming element is practically
undesorbed even while the ion exchange resin is in contact with the
amino-acid-containing solution and during the desorbing operation.
Gathering from such phenomena, the amino acid sorbed by the ion exchange
resin in this invention appears to be fixed in the ion exchange resin by a
sort of "occlusion" rather than the so-called "ion exchange". Its exact
mechanism has however not been fully elucidated.
The method of this invention can also be practiced batchwise by using a
tank-shaped vessel or the like instead of conducting the same in a
continuous manner by using a column.
According to the method of the present invention, selective sorption of one
or more target amino acid or acids is feasible without exposing the amino
acid or acids to the same strongly acidic or strongly basic atmosphere as
in the conventional ion exchange methods. Further, the target amino acid
or acids can be efficiently separated without using any chemical reagent
for pH adjustment and any acid, alkali or organic solvent as a desorbing
agent, although this has not been achieved by the prior art. The method of
the present invention is therefore suitable. The present invention has
brought about a further advantage that the resin can be used, as it is,
for the next separation treatment after completion of the desorbing
operation.
The present invention will hereinafter be described in detail by the
following examples, in which all designations of "%" are weight-basis
concentrations in the corresponding solutions. Liquid chromatography was
used for the analysis of the composition of each solution.
EXAMPLE 1
A column of 16.0 mm in diameter was packed with "Lewatit TSW40-Na", a
strongly acidic cation exchange resin (product of Bayer AG), to 500 mm
height. Warm water was then circulated through a jacket to maintain the
column at 30.degree. C.
As an amino acid solution which was a stock solution to be treated, was
employed an aqueous solution (stock solution) having the following
composition--tryptophan: 1.0%, serine: 1.0%, glycine: 1.0%, and indole:
0.1%.
Into the column, 400 ml of the stock solution were charged as a downflow at
a flow velocity of 2 in terms of space velocity. Through an outlet of the
column, 400 ml of a treated solution were obtained The treated solution
had the following composition-- tryptophan: 0.01% or less, serine: 0.98%,
glycine: 0.99%, and indole: 0.1%.
Subsequent to the above sorbing operation, 400 ml of purified water as a
desorbing solvent were charged as a downflow at a flow velocity of 2 in
terms of space velocity, and 400 ml of a desorbed solution were obtained
through the outlet of the column. The desorbed solution had the following
composition--tryptophan: 0.95%, serine: 0.02%, glycine: 0.01%, and indole:
0.001% or less.
The recovery rate of tryptophan into the desorbed solution was 95%, whereas
the exclusion rates of serine and glycine from the desorbed solution were
98% and 99%, respectively.
After the desorption with purified water, a similar sorption/desorption
operation was conducted again using the resin as it was. Substantially the
same separation results were obtained.
EXAMPLE 2
The procedures of Example 1 were repeated under the same conditions except
for the replacement of the strongly acidic cation exchange resin by
"Lewatit MDS1368-K" (product of Bayer AG). The following results were
obtained.
______________________________________
Concentrations
Concentrations
in solution
in solution
after treatment
after desorption
______________________________________
Tryptophan .ltoreq.0.01%
0.96%
Serine 0.98% .ltoreq.0.01%
Glycine 0.98% .ltoreq.0.01%
Indole 0.1% .ltoreq.0.001%
______________________________________
The recovery rate of tryptophan into the desorbed solution was 96%, whereas
the exclusion rates of serine and glycine from the desorbed solution were
both 98%.
After the desorption with purified water, a similar sorption/desorption
operation was conducted again using the resin as it was. Substantially the
same separation results were obtained.
EXAMPLE 3
Under the same column conditions as in Example 1, 400 ml of a solution
containing 1.0% of phenylalanine and 0.05% of tyrosine were treated as a
stock solution. Through the outlet of the column, 400 ml of an effluent
were obtained. The concentrations of phenylalanine and tyrosine in the
effluent were both 0.001% or less.
The recovery rates of phenylalanine and tyrosine into the desorbed solution
were both 99%.
After the desorption with purified water, a similar sorption/desorption
operation was conducted again using the resin as it was. Substantially the
same separation results were obtained.
EXAMPLE 4
Following the procedures of Example 1 except for the replacement of the
strongly acidic cation exchange resin by "Lewatit S100-Na" (product of
Bayer AG), 400 ml of a solution containing 1.0% of N-acetyltryptophan were
treated. Through the outlet of the column, 400 ml of an effluent were
obtained. The concentration of N-acetyltryptophan in the effluent was
0.001% or less.
After the desorption with purified water, a similar sorption/desorption
operation was conducted again using the resin as it was. Substantially the
same separation results were obtained.
EXAMPLE 5
The procedures of Example 1 were repeated under the same conditions except
for the replacement of the strongly acidic cation exchange resin by
"Lewatit TSW40-Ca" (product of Bayer AG). The following results were
obtained.
______________________________________
Concentrations
Concentrations
in solution
in solution
after treatment
after desorption
______________________________________
Tryptophan .ltoreq.0.01%
0.96%
Serine 0.98% .ltoreq.0.01%
Glycine 0.98% .ltoreq.0.01%
______________________________________
The recovery rate of tryptophan into the desorbed solution was 96%, whereas
the exclusion rates of serine and glycine from the desorbed solution were
both 98%.
EXAMPLE 6
The procedures of Example 1 were repeated under the same conditions except
that a cation exchange resin obtained by converting "Lewatit S100-Na"
(product of Bayer AG) into its Mg type was used in place of the strongly
acidic cation exchange resin and an aqueous solution having the following
composition--tryptophan, phenylalanine, alanine, serine and glycine: 1.0%
each--was used as an amino acid solution to be treated. The preparation of
the Mg type was conducted in the following manner. "Lewatit S100-Na" (200
ml) was packed in a column, and a 4% aqueous HCl solution in an amount as
much as 10 times the amount of the resin was caused to flow down through
the column so that the resin was converted into the H form. A 4% aqueous
MgCl.sub.2 solution in an amount as much as 10 times the amount of the
resin was then caused to flow down through the column so that the resin
was converted into the Mg-type. Thereafter, the resin was washed with 10
volumes of purified water.
The following results were obtained.
______________________________________
Concentrations
Concentrations
in solution
in solution
after treatment
after desorption
______________________________________
Tryptophan 0.01% 0.97%
Phenylalanine
0.02% 0.95%
Alanine 0.96% 0.021%
Serine 0.98% 0.01%
Glycine 0.97% 0.01%
______________________________________
The recovery rates of tryptophan and phenylalanine into the desorbed
solution were 97% and 95%, respectively, whereas the exclusion rate of
alanine, serine and glycine from the desorbed solution were 96%, 98% and
97%, respectively.
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